The programmed death ligand 1 (PD-L1) tumor expression is a manifestation of immune evasion and is used as a biomarker for treatment by immunotherapy and appears as a promising therapeutical option for Gastric Cancer (GC). The aim of this study was to evaluate the correlation between EBV status, PD-L1 expression and overall survival in a cohort of a thousand GC patients. Of the thousand tumors, 190 were EBV-positive and presented a high relative expression of PD-L1. the PD-L1 low expression was a characteristic of GC patients EBV-negative. We also show that the high expression of PD-L1 impacts on the survival probability and increase overall survival in GC patients. No EBV-negative GC patients had expression of PD-L1 protein, which suggests that the low expression is related to low overall survival. Patients with GC positive for EBV, presenting PD-L1 overexpression can benefit from immunotherapy treatments and performing the quantification of PD-L1 in gastric neoplasms should be adopted as routine.p < 0.0001) in relation to non-neoplastic control. On the other hand, in EBV-negative samples, the majority had a low PD-L1 expression of RNA and protein (p < 0.0001). In the Kaplan–Meier analysis, the probability of survival and increased overall survival of EBV-positive GC patients was impacted by the PD-L1 overexpression . However, the PD-L1 low expression was correlated with low overall survival in those patients. Patients with GC positive for EBV, presenting PD-L1 overexpression can benefit from immunotherapy treatments and performing the quantification of PD-L1 in gastric neoplasms should be adopted as routine.Gastric cancer (GC) is a worldwide health problem, making it one of the most common types of cancer, in fifth place of all tumor types, and the third highest cause of cancer deaths in the world. There is a subgroup of GC that consists of tumors infected with the Epstein–Barr virus (EBV) and is characterized mainly by the overexpression of programmed cell death protein-ligand-1 (PD-L1). In the present study, we present histopathological and survival data of a thousand GC patients, associated with EBV status and PD-L1 expression. Of the thousand tumors analyzed, 190 were EBV-positive and the vast majority (86.8%) had a high relative expression of mRNA and PD-L1 protein ( Gastric cancer (GC) is a major health problem, making it one of the most common types of cancer, in fifth place of all tumor types, and the third highest cause of cancer deaths in the world, affecting men at an average rate of two times more than in women ,2. The tFor northern Brazil, without considering non-melanoma skin tumors, GC is the second and fifth most frequent type of cancer in men and women, respectively . GC was Is widely known that tumor cells are able to express a large number of co-inhibitory immune ligands that promote an immune evasion in the tumor microenvironment that leads to progression and metastasis. Programmed death ligand 1 and 2 (PD-L1 and PD-L2) are the two ligands to the programmed cell death protein-1 (PD-1) receptor. PD-L1 is largely expressed in immune and tumor cells, while antigen-presenting cells are responsible for expressing the PD-L2 ligand. In the tumor microenvironment, a large amount of tumor-infiltrating lymphocytes (TILS) play an important role in the release of IFN-γ, and thus, induce the expression activation of PD-L1 in the tumor cells and in stromal and blood cells ,10. In tIn this scenario, the presence of PD-L1 expression in EBV-positive GC tumors can benefit those patients with immunotherapy, once testing the expression of PD-L1 is the current standard in most solid tumors: several studies have evaluated clinical results according to the status of expression of PD-L1 in GC . The aimBetween 2005 and 2015, a total of 1000 GC patients who underwent surgical resection were included in this cohort. The removed tumor samples were carefully dissected, and the tumor tissues and corresponding non-neoplastic tissues of the gastric mucosa were collected to perform a comparative study of the tumor tissue with its non-tumor correspondent, which was used as a control. That is, there were 2000 paired samples (1000 tumor and 1000 controls). The pathological staging of the GC followed the classification of tumors, nodules, metastasis (TNM) of the 8th American Joint Cancer Committee/Union for International Cancer Control (AJCC/UICC) .In previous evaluations before surgery and for tumor staging, all the patients included in this study underwent chest X-ray, abdominal ultrasound, or an abdominal computed tomography (CT) scan. Gastrectomy was performed based on the distance between the cardia and the tumor. For distal GC, the subtotal gastrectomy is the standard procedure, while for proximal GC total gastrectomy is the common procedure. Regarding the extension of lymphadenectomy, at least one dissection of gastrectomy D1  was performed for early GC, while at least one dissection D2  was applied in cases of advanced GC. For D2 dissections, a combined organ resection was sometimes performed to obtain a curative resection. All patients’ procedures followed were in accordance with Helsinki guidelines and regulations. This study was approved by the Ethics Committee of Ophir Loyola Hospital .For the patient’s follow-up and survival analysis, the assessments were performed every 3 months, after 3 years of surgery and after that period, the assessments were performed for every 6 months until the patient’s death. The follow-up procedures included the review of the following parameters: physical examinations, medical records, common blood tests, chest radiography, abdominal ultrasound, and computed tomography. In case of tumor recurrence, those were diagnosed mainly by biopsies or imaging, when biopsies were not possible to obtain. Patients with tumor recurrence were eligible to receive chemotherapy based on 5-fluorouracil (FU). All patients included in this study did not receive any previous preoperative chemotherapy.For molecular and expression analysis, biological material was conserved in liquid nitrogen shortly after surgical procedure. The DNA was extracted with phenol-chloroform, after the tumor tissue underwent a maceration procedure . For patH. pylori in all patient’s gastric samples a commercially rapid urease test  was performed according to kit instructions. The following procedures were applied before urea hydrolysis: samples were placed in a tube containing 20 g/L of Christensen’s urea agar and incubated at 37 °C for 24 h. The Polymerase Chain Reaction (PCR) in agarose electrophoresis gel was performed to confirm all negative results and to evaluate the presence of the cagA gene in H. pylori positive samples, all samples were tested in duplicate. The oligonucleotides used here have been described by Covacci and Rappuoli [To detect the presence of Rappuoli .®, Carpinteria, CA, USA) and a rabbit anti-biotinylated immunoglobulin .EBV presence in gastric tumor samples was detected using a biotinylated primer probe (5′-AGACACCGTCCTCACCACCCGGGACTTGTA-3′), which is complementary to the small EBV-encoded RNA-1 (Eber1) . For EBV®, Carpinteria, CA, USA) and the chromogen diaminobenzidine  were used to detect this reaction. The slides were contrasted with Harris’ hematoxylin and the cells were examined under light microscopy at 40× magnification. In this examination, 10 representative microscopic fields containing at least five cells were evaluated. The epithelial cells were considered positive for EBV presence when 5% or more cells were stained in brown/red. Both tumor and non-neoplastic samples from the same patient were analyzed.The streptavidin-biotin peroxidase complex , according to the manufacturer’s protocol. The RNA conversion into cDNA was performed using the High Capacity® cDNA Reverse transcription kit , following the manufacturer’s instructions. The cDNA was amplified by quantitative real-time PCR (RT-qPCR) using TaqMan probes  and amplified on a 7500 Real-Time machine . All RT-qPCR sample reactions were analyzed in triplicate. The cycle threshold (Ct) and expression values with standard deviations were calculated. The GAPDH gene was selected as an internal control for RNA entry and reverse transcription efficiency. All RT-qPCRs were performed in triplicate for the target gene (PD-L1 Hs01125297_m1) and the internal control (GAPDH: NM_002046.3).The total mRNA of the 1000 GC patients was isolated from tumor tissues and their paired adjacent non-tumor tissues (confirmed by pathology as non-tumor). RNA was obtained using Tri-reagentThe gene expression relative quantification was calculated according to the guidelines . The conThe total proteins of the paired samples (tumor tissues and non-neoplastic tissues) were extracted using the M-PER regent for the extraction of mammalian proteins . The purified proteins of samples were separated by SDS-PAGE and electrotransferred on a PVDF membrane  for detection and expression. The PVDF membrane was blocked with phosphate-buffered saline containing 0.1% Tween 20 and 5% low-fat milk and incubated overnight at 4 °C with the corresponding primary antibodies: anti-PD-L1  and anti-beta actin .After several washing steps with TBS, a secondary antibody conjugated to peroxidase was added for 1 h at room temperature. The immunoreactive bands were visualized using the Western blotting reagent Luminol, and the images were acquired using an ImageQuant 350 digital imaging system . Beta-actin (ACTB) was used as a loading control and the relative quantification of protein expression was calculated following the same basis as the quantification of mRNA, that is, a control sample of non-neoplastic gastric tissue adjacent to the analyzed tumor, was designated as a calibrator for each tumor.2 test and Fisher’s exact test were used to assess continuous variables. The data are shown as the median and interquartile range (IQR) depending on the normality of the samples, the Shapiro–Wilk test was applied to determine whether the samples followed a normal distribution. For comparison analysis between two or more different groups, corresponding nonparametric tests were applied. Linear regression was calculated with the ggpubr R package and were considered significant with p < 0.05. In the quantification of mRNA and PD-L1 protein, they were only considered significant when these two biomarkers simultaneously had p-values < 0.05.Pearson’s chi-square χhttps://www.r-project.org, accessed on 20 March 2021).The overall survival of patients was calculated from the date of diagnosis to the date of death or the last follow-up assignment, using the Kaplan–Meier method with the log-rank statistical. All graphs and statistical analysis were done by using R (p < 0.0001) and the number of female patients (34.2%) was statistically lower (p < 0.0001) than the number of male patients (65.8%). The number of patients presenting with the initial disease, in stage I (14.3%), was statistically lower (p < 0.0001) than all stages, mainly stage IV, the most advanced (38.3%).GC samples were collected from 1000 patients between 2005 and 2015. The average age was 58 years . The number of young patients, under 64 years of age, was statistically higher (61.8%) than patients older than 64 years of age (p < 0.0001) between the diffuse (41.2%) and intestinal (58.8%) subtypes. Regarding tumor location, tumors located in cardia were significantly more frequent (p < 0.0001) than others in different locations in the stomach. There was no statistical difference between the number of patients with and without metastases in other organs. Lauren’s classification  also shoH. pylori and EBV, we can observe that the presence of the bacterium was significant (p < 0.0001): 82.8% of the patients presented with infection by H. pylori, and 61.9% (p < 0.0001) of the patients had the virulence conferred by the presence in the genome of the bacterium of the cagA gene. In the case of EBV infection, the absence of the virus in most of the studied samples is significant (p < 0.0001), only 19.0% of the studied samples had the viral genome (Regarding infections by l genome .p < 0.0001) in relation to the non-neoplastic control. Regarding protein expression, 149 (78.4%) had high expression and 41 (21.6%) low protein expression (p < 0.0001) In the EBV-negative samples, the vast majority had RNA expression and low PD-L1 protein (p < 0.0001) (p < 0.0001)  had high relative PD-L1 mRNA expression, and 25 tumors had low PD-L1 mRNA expression (13.2%) ( 0.0001) , Table 2p < 0.0001) than the expression profiles of the intestinal type. In the analysis of patients with EBV-negative status, those of the diffuse and intestinal-type do not have a statistically significant expression of mRNA and protein (p > 0.05) (In  > 0.05) A,B.There is a linear correlation of the increase in mRNA and PD-L1 protein levels in EBV-positive patients with a diffuse histological type (R = 0.90) and intestinal-type (R = 0.86) (p < 0.0001), with diffuse histological type tumors located in the cardia (p < 0.0001) and infected with H. pylori cagA positive (p < 0.05). EBV-negative patients often have metastases in other organs and low expression and production of PD-L1 (p < 0.001)  D. The cop < 0.05) in stage I/II tumors, with no lymph node metastases (N0) and in other organs (M0) (p < 0.001). Overexpression was significant in tumors of the diffuse subtype (p < 0.05), located in cardia (p < 0.001). There was no statistical association with the presence or absence of H. pylori cagA positive bacteria. These results are summarized in The high expression of PD-L1, regardless of EBV infection, was significantly higher (In p < 0.001) than intestinal tumors, although both histological types are over-expressed for PD-L1 when infected with this virus.The presence/absence of lymph node and other organ metastases in EBV-negative patients is not associated with the production of PD-L1, whereas EBV-positive samples have a higher production of PD-L1 in samples from patients without lymph node metastasis or in other organs. Regarding the histological type, EBV-negative patients do not show differences in the production of PD-L1, always remaining below 1.5 or 50%. On the other hand, samples of diffuse EBV-positive tumors produce greater expression of PD-L1 (p < 0.001) in EBV-positive patients (p < 0.0001). The levels of mRNA and protein of PD-L1 stratified in samples obtained from different locations of the stomach are significantly higher in EBV-negative patients whose tumor is located in the gastric pylorus (p < 0.001) in relation to the other locations . However, the low expression of PD-L1 accentuates the low overall survival in patients with diffuse GC more than in the intestinal histological type.The overall survival (in months) of GC patients with diffuse and intestinal histological types were analyzed by Kaplan–Meier curve. In The expression data are described in p = 0.0039). In p < 0.0001). When comparing stages I/II with III/IV .The comparison of the overall survival (in months) between patients with diffuse and intestinal histological GC performed by Kaplan–Meier analysis are shown in n = 302) [EBV can directly cause the development of GC, and the prevalence of EBV associated with GC varies from 4 to 18%, with a frequency twice as high in males . The incn = 302) , this inn = 302) ,25,26. In = 302) ,28,29, tEBV shows an anatomical preference during gastric tumorigenesis and is predominantly present in the proximal stomach, with reported rates of 11.6% in the cardia and 9.5% in the stomach body ,30. A Kop < 0.001). Regarding metastases in other organs, we can observe a significantly lower number of cases associated with EBV with metastases than EBV-positive patients with no metastases , in stage IV cases, the latter finding can be explained because the public hospital where the samples were obtained serves patients belonging to a more needy population in the region, which normally seeks hospital services in advanced stages of cancer, and therefore, a greater number of advanced cases is always expected.In addition, the overall survival analysis (at 60 months) of GC patients clearly shows a higher survival of EBV-positive patients when compared to EBV-negative patients. Although the number of EBV-negative cases was found to be significantly higher using Fisher’s exact test (p < 0.001), in this study  described the PD-L1 expression as a negative prognostic factor for overall survival, and only three studies associated it as a positive prognostic factor, with one study reporting no prognostic significance . Howeverp < 0.0001). Another study with a large number of samples (n = 3241) also found results similar to our findings, EBV-positive patients (5%) were young and male, with GC samples histologically poorly differentiated, and patients having a good prognosis when expressing the PD-L1 protein [Our findings are also corroborated by the study by Moore et al. , who fou protein . The dif protein ,22.PD-L1 protein expression is used as a prognostic biomarker for treatment by immunotherapy. Immunotherapy appears as a promising novel treatment for patients with metastatic or recurrent GC. The PD-L1 tumor expression is described as a manifestation of immune evasion. As mentioned, the distinctive characteristic of GC positive for EBV, in relation to EBV-negative, is the overexpression of PD-L1. The positivity of PD-L1 in GC positive for EBV can be effectively directed towards immunotherapy; testing the expression of PD-L1 should therefore become routine in GC ,42.Immunotherapy based on PD-L1 inhibitors are an important innovation in tumor therapy in recent years, which can reactivate T cell immune functions, increase the body’s immune response capacity and, ultimately, allow the immune system to eliminate gastric cancer tumor cells effectively and achieve tumor remission. Inhibition of the programmed death-1 (PD-1)/programmed-ligand 1 (PD-L1) axis with immune control point inhibitors (ICPI), including nivolumab, avelumab and pembrolizumab, has emerged as new therapeutical strategy for GC patients. Treatment with ICPI in GC provides a survival benefit and anti-PD-1 treatment can especially improve the overall survival rate and prolong the duration of the response. In addition, anti-PD-1/PD-L1 therapy is likely to be more effective in subgroups with high microsatellite instability, EBV-positive, or high mutation load in advanced GC ,43.The targeted therapy scenario for GC is promising, and many clinical trials evaluating immune response to those therapies combined with targeted molecular agents are generating a great deal of enthusiasm. In addition, genomic data from The Cancer Center Genome (TCGA) and Asian Cancer Research Group (ACRG) classifications are being widely explored to identify molecular features of tumor subtypes to allow, in the near future, clinical trials that might include GC patient populations enriched with distinct biomarkers .GC molecular classification has emerged as an important tool to select the right patients who will benefit from those targeted therapies or benefits of an accurate disease prognosis. The addition of the new molecular classification strategy to a current classic subtype can be a promising option, particularly EBV virus stratification. The findings of predictive biomarkers, such as PD-L1, should improve patient diagnosis and therapy in the era of personalized medicine, once targeted therapies can potentially change the GC patients’ treatment scenario, leading to a better understanding of heterogeneity and better results.Patients with GC positive for EBV and presenting PD-L1 overexpression can benefit from immunotherapy treatments and performing the quantification of PD-L1 in gastric neoplasms should be adopted as routine.


Background: Mirror therapy is thought to drive interhemispheric communication, resulting in a balanced activation. We hypothesized that embodied virtual mirror visual feedback (VR-MVF) presented on a computer screen may produce a similar activation. In this proof-of-concept study, we investigated differences in movement-related cortical potentials (MRCPs) in the electroencephalogram (EEG) from different visual feedback of user movements in 1 stroke patient and 13 age-matched adults.Methods: A 60-year-old right-handed (Edinburgh score >95) male ischemic stroke  patient and 13 age-matched right-handed (Edinburgh score >80) healthy adults  participated in the study. We recorded 16-electrode electroencephalogram (EEG), while participants performed planar center-out movements in two embodied visual feedback conditions: (i) direct  and (ii) mirror  with left (direct left/mirror left) or right (direct right/mirror right) arms.Results: As hypothesized, we observed more balanced MRCP hemispheric negativity in the mirror right compared to the direct right condition . MRCPs in the stroke participant showed reduced lateralized negativity in the direct left (non-paretic) situation compared to healthy participants. Interestingly, the potentials were stronger in the mirror left (non-paretic) compared to direct left case, with significantly more bilateral negativity at FC3 [95% CI (0.758 13.2)] and C2 [95% CI (0.04 9.52)].Conclusions: Embodied mirror visual feedback is likely to influence bilateral sensorimotor cortical subthreshold activity during movement preparation and execution observed in MRCPs in both healthy participants and a stroke patient. Post-stroke motor rehabilitation techniques, such as constraint-induced movement therapy , requireVirtual reality (VR) applications are emerging in post-stroke motor rehabilitation procedures . VR may The Human Research Ethics Committee of the Canton of Vaud, Switzerland, approved the study. The study was conducted in the Acute Neurorehabilitation Unit of the Centre Hospitalier Universitaire Vaudois (CHUV) in Lausanne, Switzerland. All participants provided written informed consent before the experiment.A cohort of 13 healthy right-handed volunteers  was recruited for this study.Outcome measures) were obtained on day 8 , day 13 , and day 706 (follow-up T3). The patient performed the VR task on three consecutive days, namely, days 9, 10, and 11, using the MindMotionTM PRO VR system  at the patient's bedside in the acute neurorehabilitation center. The patient also received ~60 min of full-body occupational therapy during the hospital stay.We recruited a 60-year-old right-handed (Edinburgh score >95) male subject who survived an ischemic stroke (left paramedian pontine) of atheromatous origin, which caused hemiparesis of the right side. The participant was hospitalized on day 0, and assessments  VR rehabilitation platform. The participants sat in a comfortable chair or wheelchair in front of a table as shown in TM PRO system consists of a motion-capture camera (placed at a height of ~180 cm) for tracking the upper extremity. In addition, a touch screen for the therapist to set up the training session and a monitor (placed at ~120 cm from participant) to display the embodied feedback via an avatar that reproduces participants' movements in real time were used. In a typical trial, participants first placed a hand on the Start pad, when ready. After a random time (maximum of 2 s), a target (35 cm away) was exposed at one of the five locations (each 32.5° apart), as described in Participants were asked to perform upper limb center-out reaching movements in a 2-dimensional plane  to five locations randomly presented using the MindMotiondirect or mirror feedback in three conditions as described in direct left, the participant moves the left arm, and the avatar reproduces the movement with its left arm; (b) mirror right, the participant moves the right arm and the avatar reproduces the movement with its left arm; and (c) direct right, the participant moves the right arm and the avatar reproduces the movement with its right arm. Each participant performed ~100 repetitions per condition per session with conditions presented in blocks (25 repetitions) of a random order.Healthy participants performed the planar center-out reaching movements with direct left ; (b) mirror left, the participant moves the left arm (non-paretic), and the avatar moves its right arm ; and (c) direct right, the participant tried to use the right arm (paretic) with 10, 85, and 90 repetitions on days 9, 10, and 11, respectively. The order of the conditions was chosen according to the patient's comfort, where we started with direct right condition and shifted to other conditions whenever the patient reported fatigue in the paretic arm, allowing the arm to recover.The stroke participant performed the center-out reaching tasks over three consecutive days , bottom,g.USBamp recording system  for all participants. The EEG data was sampled at 512 Hz and synchronized with MindMotionTM PRO exercise events that were detected using a 3D motion-capture system.We recorded full-band EEG (fbEEG) in 10–20 international system with 16 electrodes  using the Stroke participant).We measured the patient's stroke severity using the National Institutes of Health Stroke Scale (NIHSS), motor impairment using the Fugl–Meyer Assessment for Upper Extremity subscale (FMA-UE) , and motWe performed data processing using Matlab R2013a .fc = 25.6 Hz). After this, the data were band-pass filtered in the range of 0.1–1.5 Hz corresponding to the spectral content of slow cortical potentials (SCPs)  . For heas (SCPs) ]. For thdirect right (paretic arm) after artifact rejection, we limited further analysis to direct left and mirror left conditions. We produced topographic plots of the average activity at each channel over a 25-ms time window, selected using the time point that corresponded to the maximal difference between the observed conditions.Trials were extracted using a −2 to +2 s time window around movement onset at zero seconds . For eacdirect left, direct right, or mirror right conditions. The observations consisted of the pooled data for all participants using the mean activity corresponding to the same temporal window as the topographic map described in Electroencephalogram grand averages. We then used Tukey's test for pairwise differences in direct right vs. direct left and direct right vs. mirror right. Similarly, for the stroke participant's data, we performed Tukey's test for each electrode separately to assess the differences between direct left (non-paretic) and mirror left (non-paretic) conditions.For healthy participant data, we used ANOVA to assess differences in the negativity of MRCPs between electrodes depending on their location. We used a single, three-leveled factor: direct left, direct right, and mirror right conditions. Like the well-known readiness potentials, we observe an increase in negativity for all the three conditions with respect to the baseline period, however with spatial differences described below. (i) Direct left and direct right showed higher negative activity on the contralateral side, i.e., right  and left-side electrodes  compared to the ipsilateral electrodes, respectively. (ii) The contralateral negativity is highest for the direct left condition, with peak negative activity at the C2 electrode (mean ± SEM: −18.0 ± 1.0 μV at 0.4 s), negativity at the Cz electrode (−17.0 ± 1.1 μV at 0.4 s), and negativity at the C1 electrode (−14.5 ± 0.75 at 0.4 s). For the direct right condition, we observe similar higher negativity at the contralateral electrode  compared to the ipsilateral electrode maximal negativity , but with the highest negativity at the vertex electrode . (iii) Interestingly, the mirror right condition showed bilateral negativity at the central electrodes, such as C1 (−14.0 ± 0.8 at 0.25 s), Cz (−14.8 ± 0.8 at 0.30 s), and C2 (−10.0 ± 0.7 at 0.30 s).The grand-averaged traces for the pooled data of healthy participants are presented in direct right condition displayed negativity at the left-side electrodes  at electrodes FC3 (F = 7.98), FCz (F = 18.7), FC4 (F = 33.4), C3 (F = 12.6), Cz (F = 21.6), C2 (F = 29.9), C4 (F = 41.7), CPz (F = 31.5), CP2 (F = 31.3), and CP4 (F = 32.4). Pairwise tests between direct right and direct left revealed significant differences (p < 0.01) at fronto-central, central, and centro-parietal electrodes . Similarly, a significant difference between direct right and mirror right was observed at the FC4 electrode .We performed one-way ANOVA at the group level for the condition factor . The results revealed significant differences (direct left (non-paretic) and mirror left (non-paretic) conditions. In the direct left (non-paretic) condition, we observed negative potentials at the central electrodes, but with less clear lateralization in the maximal negative activity . Interestingly, the mirror left (non-paretic) condition elicited similar patterns yet with higher negativity . Overall, the appearance of the negative peak was earlier than that of the healthy participants.We present the grand-average MRCPs of the stroke participant in mirror left (non-paretic) and direct left (non-paretic) conditions on both the ipsilesional {FC3 electrode [95% CI ] and contralesional sides [C2 electrode 95% CI ].We show topographic maps of MRCP activity in The NIHSS score decreased from six at pre-intervention (T1) to three at discharge (T2), corresponding to a mild impairment . The scoThe present study explored the nature of MRCPs due to MVF delivered using an embodied visual feedback on a computer screen during reach movements in a stroke participant and age-matched adults. In healthy participants, we reported MRCPs with peak activities in the hemisphere contralateral to the active arm in direct left and direct right situations, as expected , 15. Intmirror left condition, we found stronger and bilateral (symmetric) activity at the central electrodes, which is in line with the healthy participant data. This is likely due to the interhemispheric communication through mirror feedback  condition showed less lateralization with similar negativity patterns at C1 and Cz electrodes. The recruited patient presented a pontine lesion, i.e., the anatomical pathways mediating interhemispheric interactions were preserved, a condition not always possible in patients with stroke. Thus, this difference could be due to natural individual differences or being stroke specific, which should be confirmed with more data of different stroke profiles in different phases of the recovery. We also observed a reduced latency of the negative peak compared to healthy participants, which has previously been reported in the literature . In the feedback , which mdirect right (using the paretic arm), 210 direct left (using the non-paretic arm), and 215 mirror left (using the non-paretic arm) repetitions, which represents high dosage during the acute hospitalization period. Pre- and post-VR task motor assessment showed an increase in the FMA-UE score of 6 points, which is more than the minimal clinically important difference in FMA-UE score (It is worth noting that over the three intervention days (days 9–11), the patient performed 185 UE score . At follUE score  showed tUE score  reportedCalabrò et al.  showed tIn the current proof-of-concept study, we report that cortical excitability is likely modulated using VR-MVF in both healthy participants and a stroke patient and hypothesize that such an intervention may potentially activate neural mechanisms involved in stroke recovery.The original contributions presented in the study are included in the article/supplementary material. Further inquiries can be directed to the corresponding author/s.The studies involving human participants were reviewed and approved by The Human Research Ethics Committee of the Canton of Vaud, Switzerland (CER-VD). The patients/participants provided their written informed consent to participate in this study.GG and DP-M contributed to project design and its conception and writing the first draft of the manuscript. TR performed data analysis. JJ contributed to the practical execution of the study. KD was the principal investigator of the study and contributed to planning and overseeing the study. All authors contributed to the manuscript revision, read, and approved the submitted version.GG and DP-M were employees at MindMaze SA, Lausanne, Switzerland at the time of the study. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The study evaluates the interaction of BIDs through molecular docking simulations, molecular dynamics simulations, and binding free energy calculations. This is the first ever computational approach to discover the potential inhibitors of presenilin proteins. It also comprises druglikeliness and pharmacotherapeutic potential analysis of the compounds. Out of all the screened BIDs, BID-16 was found to be the lead compound against both the presenilin proteins. Based on these results, one can evaluate BID-16 as an anti-Alzheimer’s potential specifically targeting presenilin proteins in near future using in vitro and in vivo methods.The most commonly accepted hypothesis of Alzheimer’s disease (AD) is the amyloid hypothesis caused due to formation of accumulation of Aβ42 isoform, which leads to neurodegeneration. In this regard, presenilin-1 (PSEN-1) and -2 (PSEN-2) proteins play a crucial role by altering the amyloid precursor protein (APP) metabolism, affecting γ-secretase protease secretion, finally leading to the increased levels of Aβ. In the absence of reported commercial pharmacotherapeutic agents targeting presenilins, we aim to propose benzophenone integrated derivatives (BIDs) as the potential inhibitors of presenilin proteins through  It is the most common type of dementia found in nearly 60–70% of the cases according to the WHO report. According to the Alzheimer’s association report, mortality rate has been increased by 146.2% between 2000 to 2019, with nearly 44 million people globally suffering till date , 2. Based on the recent clinical evidence, it is known that the progression of Alzheimer’s disease is due to the formation of beta-amyloid plaques, which are formed by the cleavage of amyloid precursor protein (APP) by β-secretase and γ-secretase protease enzymes . The Aβ42 oligomer isoform is the major toxic constituent in neurodegeneration AD. The toxic Aβ oligomers interact with glial cells causing inflammatory cascades, oxidative stress due to increased production of reactive oxygen species (ROS) and reactive nitrogen species (RNS). This causes the loss of function in many antioxidant defence enzymes, dysfunction in calcium metabolism which together leads to neuron degradation. Furthermore, these events cause dysfunction in APP metabolism leading to Aβ neurotoxic peptides causing AD , 5.The pathological cause for Alzheimer is the formation of extracellular amyloid plaques (Aβ) and cytoplasmic neurofibrillary tangles (NFT’S). The protein fragments of beta-amyloid accumulates outside the neurons, whereas tau protein accumulates inside them . After mapping of the PSEN-1, it was found that PSEN-2 with 60% homology to that of PSEN-1 was found on chromosome 1q42.13 encoding its γ-secretase subunit responsible for the development of AD . The PSEN-1 plays a crucial role in the pathogenesis of AD as it is found with over 300 mutations . Recently, it is reported that both PSEN-1 and PSEN-2 are the functional subunits of γ-secretase . As per the study conducted by Silveyra et al. (2008) , the correlation between PSEN-1 and AChE indicates that PSEN-1 can physically interact with AChE with little understanding on its mode of interaction. According to Belluti et al. (2014) , benzophenones-based derivatives can be a new promising target for AD, which inhibit AChE at sub-micromolar level.From recent advances in research, it has become evident that PSEN-1 gene, which is located on chromosome 14q24.2 and encodes PSEN-1 protein subunit of γ-secretase, is the main cause for early-onset of AD –14. In this work, we investigate the interaction of the benzophenones derivatives by using in silico approach using computational tool to analyse and elucidate the biological property of benzophenones derivatives with the Presenilin (PSEN) proteins. So far, this is the first in silico investigation on the inhibition of PSEN proteins.Benzophenones are a group of compounds with more than 300 biologically active members with a common phenol-carbonyl-phenol skeleton and excellent structural diversity. These derivatives obtained from natural and/or synthetic sources are biologically active molecules displaying antitumor, anti-inflammatory, and antidiabetic activities. Several analogues of benzophenones are under clinical study; Combretastatin A-4 is well-known to exhibit antiangiogenic effects and is being studied in clinical trials. Benzophenones associated with other heterocyclic analogues possess potential applications rather than benzophenone alone. In this context, to pursue a potential approach towards the discovery of most efficient pharmacologically active compounds, the benzophenones are amalgamated with a new class of heterocyclic compounds. The presence of indole, oxadiazole, and benzimidazole nucleus with benzophenone in numerous categories of therapeutic agents has made it an essential anchor for the development of new pharmacophore https://www.uniprot.org/) for which template search was completed using BLAST tool to find the best template. The Human gamma-secretase in complex with small molecule avagacestat (PDB: 6LQG) which had sequence identity of 100% for PSEN-1 and 71.23% for PSEN-2 was selected. The sequence was aligned to the template protein and the models were built using SWISS-MODEL (https://swissmodel.expasy.org/) . The structures were energy computed using GROMOS96 43B1 parameter using SWISS PDB Viewer . Further, the modelled structures were validated based on Ramachandran plot Figs  and 2 to get a better understanding of the structure compatibility.The protein sequences of PSEN-1 (ID: P49768-1) and PSEN-2 (ID: P49810-1) were obtained from UniProt ((http://sts.bioe.uic.edu/castp/index.html?3trg)  server is used. The server identifies and measures the volume and area of all the available binding pockets. The program was used to get the binding site of PSEN-1 and PSEN-2 proteins.To know the topographic feature and to get the better understanding of the binding pockets of the protein, Computed Atlas of Surface Topography of proteins (CASTp) , which uses Broyden-Fletcher-Goldfarb-Shanno algorithm to get a better insight of the conformational changes and to understand their interaction with the compound within the specified active site region. The pre-docking preparation for both proteins and ligands was done according to Autodock 4.2  protocol using Autodock Tools 1.5.6, by removing all the water molecules and hetero atoms present in the proteins. Further, non-polar hydrogens and carbon atoms were merged to obtain the stability. The Kollmann united and Gasteiger-Marsili empirical atomic partial charges that were predicted by the Autodock Tools 1.5.6 were assigned to balance the structure. Similarly, the torsions were kept as default as given by the program, AD4 atom type were added to determine the types of atoms in the macromolecule, and the prepared structures were obtained in PDBQT format with inclusion of charges Q and AutoDock 4 atom types T for molecular docking simulation.Molecular docking for the modelled PSEN-1 and PSEN-2 protein with the compounds was performed using AutoDock Vina 1.1.2 Based on the CASTp server prediction, the grid box for the binding pockets was prepared. For PSEN-1, the grid box of size x = 38.16 Å, y = 38.16 Å, z = 38.16 Å were set centred to cover the binding pockets and all essential residues at x = 174.73 Å, y = 180.58 Å, and z = 144.29Å. For PSEN-2 grid box of size x = 26.43 Å, y = 26.43 Å, z = 26.43 Å were set centred to cover the binding pockets and all essential residues at x = 162.88 Å, y = 178.42 Å, z = 152.74 Å. For the docking process, the ligand was considered to be flexible while protein was kept rigid. The binding analysis for PSEN-1 and PSEN-2 was performed using Biovia Discovery Studio Visualizer 2021 for the best docked conformation based on their binding affinity, total number of non-bonding interactions, and their hydrogen bonds. for the duration of 100 ns, to which CHARMM36 force field was applied to get the parameter for protein whereas, CGenFF python script  was used to generate the topology and force field parameter file for the ligand. Using TIP3 water model which describes the 3-site rigid water model with charges and Lennard-Jones parameter, a solvent box was created with 10 Å distance. By using genion tool the system was neutralized by maintaining the proper salt concentration of 0.15 M counter ions were added such as Na+ and Cl-. The initial energy minimization step is done for 5000 steps using steepest descent algorithm. Further, the system equilibration is done using NPT and NVT ensemble class with a 310K temperature and 1 bar pressure. The simulation was run for 100 ns with relaxation time one ps time. After simulations the trajectories were analysed using GROMACS plugin to know the stability of protein and protein-ligand complex system by analysing the geometrical properties, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), Radius of Gyration (Rg) and SASA (Solvent Accessible Surface Area) were calculated. The plot for the above trajectories was plotted using XMGRACE software .Molecular dynamics (MD) simulations were performed for the predicted docked complexes of BID-16 with PSEN-1 and PSEN-2, which showed the lowest binding affinity with least RMSD score. Based on the results of molecular docking simulation, BID’s with inferior binding affinity, less number of non-bonding interactions, and hydrogen bonds were selected as negative controls. In case of PSEN-1, BID-19 was selected as a negative control, whereas in case PSEN-2, BID-20 was selected as the same. Molecular dynamics simulation study is done to get the clear insight of the structure and thermodynamics at atomic level, stability, behaviour, and conformational changes of both the protein and the ligand molecule. MD simulation was carried out at nanosecond scales for the docked complex of BID-16 using GROMACS-2018.1  which works using GROMACS trajectories. In g_mmpbsa tool the binding free energy is calculated using three components which are molecular mechanical energy, polar and apolar solvation energies. To calculate the binding free energy, the MD trajectories of last 50 ns were considered to compute ΔG with dt 1000 frames. The binding free energy is calculated using below two Eq  approach by using g_mmpbsa program w two Eq .MM is the average potential energy in vacuum, Gsol is the sum of the solvation free energy. Further in Eq (polar and Gnonpolar represents electrostatic and nonelectrostatic support to the solvation free energy.Where Eer in Eq  is givener in Eq  is givenin silico approach using SWISS ADME (http://www.swissadme.ch/) and OSIRIS property explorer (http://www.cheminfo.org/Chemistry/Cheminformatics/Property_explorer) . The druglikeliness was evaluated based on “Lipinski’s rule of five”. Whereas, the toxicity was evaluated based on mutagenic, tumorigenic, irritant, effects on the reproductive system, drug-induced liver injury (DILI) and cytotoxic parameters.The ADMET screening was carried out to assess the druglikeliness, pharmacokinetics, and toxicity parameters of ligand molecules through , which depicted the stability, with the instability index of 36.89 for PSEN-1 and 43.45 for PSEN-2. Further, the Ramachandran plot was used to evaluate the quality of the modelled structure based on the Phi and Psi distributions. For PSEN-1 91.5% of the residues were present in favoured regions and 2.30% of them were in outlier regions . The same pattern was observed with PSEN-2, with 92.7% of the residues present in favoured, and 1.64% of the residues in outlier regions . The overall quality of Ramachandran plots was satisfactory for both the modelled structures. Tables 1 and 2 depict the physiochemical properties of PSEN-1 and PSEN-2 protein models, respectively.The modelled structures were validated for its physiochemical properties using Protparam  are given in the Figs 7 and 8, respectively.The molecular interaction of BID-16 bound PSEN-1 had total of 12 non bonded interactions, LEU B: 383 (2.94 Å), GLY B: 384 (2.03 Å), ASP B: 385 (3.40 Å), ASP B: 385 (3.75 Å) were bound with hydrogen bonds> In addition 8 hydrophobic bonds were formed, including 2 Pi-sigma bounds with LEU B: 268 (3.93 Å), LEU B: 286 (3.99 Å), 4 alkyl bonds with ALA B: 285 (4.04 Å), ALA B: 434 (4.35 Å), ILE B: 213 (4.39 Å), ILE B: 229 (4.36 Å), and 2 Pi-alkyl bonds with ILE B: 387 (5.06 Å) and ALA B: 285 (4.82 Å). On other hand, BID-16 bond PSEN-2 had total of 15 non bonded interactions, with a total of 2 hydrogen bonds with ALA B: 415 (2.26 Å), PRO B: 414 (3.34 Å), 4 electrostatic bonds were formed with ASP B: 263 (5.36 Å), ASP B: 366 (2.89 Å), ASP B: 263 (4.91 Å), ASP B: 366 (3.23 Å). A total of 6 hydrophobic bonds were formed, including a lone Pi- Pi bond with PHE B: 289 (5.16 Å), 2 alkyl bonds with LEU B: 274 (4.28 Å), LEU B: 156 (4.96 Å), and 3 Pi-alkyl bonds with LEU B: 292 (5.10 Å), LEU B: 274 (5.31 Å) and LEU B: 292 (5.46 Å). In case of negative controls, BID-19 formed a total of 9 non-bonding interactions with 1 hydrogen bond with GLY B: 382 (3.74 Å) of PSEN-1. The hydrophobic interactions included pi-sigma bonds with ILE B: 143 (3.41 Å), LEU B: 268 (3.86 Å), and LEU B: 286 (3.63 Å). It formed 3 alkyl bonds with LEU B: 383 (5.19 Å), ILE B: 387 (5.00 Å), and ILE B: 143 (4.53 Å) of PSEN-1. In addition, 2 pi-alkyl bonds with PHE B: 388 (5.40 Å) and LEU B: 286 (4.65 Å) were also formed. With these interactions, the binding affinity of BID-19 was found to be -7.6 kcal/mol. In case of PSEN-2, the BID-20 formed a total of 3 non-bonding interactions with no hydrogen bonds. It formed a hydrophobic bond with LEU B: 274 (3.53 Å), and 2 pi-alkyl bonds with ILE B: 288 (4.89 Å) and ILE B: 293 (5.33 Å). With these interactions, the binding interaction of BID-20 is -8.2 kcal/mol. The visualization of molecular docking for PSEN-1 and PSEN-2 with BID’s has been given in The Lipinski’s rule of five is used to determine the pharmacokinetic potential of the chemical compounds through the means of their absorption, distribution, metabolism, elimination (ADMET) properties. During this analysis, the drug score predicted by OSIRIS for BID-16 was 0.76. Further, the pharmacokinetic properties were predicted with the molecular weight 432.32 g/mol, number of hydrogen bond acceptors 7, number of hydrogen bond donors 2, number of rotatable bonds 7 and cLogP 3.18. There were no toxicity issues like mutagenicity, tumerigenicity, irritability reported with BID-16. Furthermore, it was predicted with no inhibition of P-glycoprotein (P-gp) substrate, cytochrome P (CYP). In case of negative controls, the druglikeliness of depict that the molecular weights of BID-19 and BID-20 is found to be 477.63 g/mol and 522.08 g/mol, respectively. Both the compounds had same hydrogen bond acceptors (3), donors (0), and rotatable bonds (6). However, the cLogP value BID-19 (6.07) and BID-20 (6.18) differed. Regarding pharmacokinetic properties, tumerigenicity was found in case of BID-19 as well as BID-20. Both of the compounds didn’t pass druglikeness parameter (-2.95 for BID-19) and (-2.45 for BID-20), as both of them violate the Lipinski’s rule of five.The molecular dynamics simulation was performed to evaluate the conformational changes and dynamic stability of the docked complexes. The MD was performed for homology models of PSEN-1 and PSEN-2 with the selected compound of BID-16 as their ligand. The simulation was carried for 100 ns to understand the interactions between the docked complexes and the results were evaluated based on, RMSD, Rg, RMSF, SASA and ligand hydrogen bond plots.. The PSEN-1 protein (green) increased up to 0.6 nm at ~5 ns and there was a slight fluctuation around 50 to 55 ns at 0.62 nm. Later, it demonstrated stability over ~78 to 80 ns and a slight dip of 0.58 ns at 80 ns, whereas protein-BID-16 complex (black) increased at a time interval of 0.5 ns at 0.55 nm. Further, there a slight rise at ~10 ns of 0.59 ns was observed that remained stable throughout the run. However, protein-BID-19 complex became stable after 90 ns. It rose to 0.8 nm at 5 ns, and the peak value (0.9 nm) was observed at 20 ns, 50 ns, and 70 ns. The whole RMSD plot of protein-BID-19 complex was found to be unstable throughout the simulation, in comparison to the protein-BID-16 complex. On the other hand, PSEN-2-BID-16 complex attained stability around 90 ns at 0.98 ns . The PSEN-2 (green) increased up to 1 nm at around 45 ns time and remained its stability throughout the simulation, whereas the protein-BID-16 complex (black) showed a slow increase up to 0.98 nm at ~35 ns, then fell to 0.9 nm at the time interval of 50 ns and remained stable through 100 ns run. The protein-BID-20 complex however, showed constant fluctuations throughout the simulation run. The peak of RMSD plot was observed at 40–45 ns (1.5 nm). Unlike protein-BID-16 complex, it was not stable from the beginning to the end.The RMSD was evaluated to know the structural stability of both the protein and ligand complex and to examine their dynamic nature during the 100 ns simulation. The values were evaluated based on the plot of PSEN-1 and PSEN-2 with BID-16. The RMSD of PSEN-1-BID-16 complex that covered during 100 ns MD simulation showed that the complex attained stability after 80 ns Figs 9B and 10B, respectively. The RMSF plot of PSEN-1 model showed a highest fluctuation at T-terminal for the residues at the 450 position at 1.5 nm, whereas the protein-BID-16 was also predicted with the same value. The protein-BID-19 complex was found with unusual fluctuations at 250–400 residues, indicating its relative instability. It was also found with highest RMSF value at T-terminal (1.5 nm). Conversely, PSEN-2 showed a highest fluctuation at loop regions 100–150 and 400 residues. Otherwise, it was found with minimal fluctuations. The protein-BID-16 complex was also found with minimal fluctuations, except loop regions. However, the protein-BID-20 complex was found with higher number fluctuations at loop regions compared to protein backbone atoms and protein-BID-16 complex, indicating its instability. It showed the highest fluctuation at 300–350 residues.The RMSF plot was analysed to evaluate the residue flexibility of complex along with the modelled protein. The RMSF was evaluated for both PSEN-1 and PSEN-2 along with the BID-16 complex as depicted in the  was 2.24 nm, followed by a slight reduction in the fluctuation value at 2.24 nm at 40 ns and showed relatively stable values thereafter . The protein as well as the complexes showed almost similar and compactable values for PSEN-1-BID-16 complex. Based on the above evaluation, it can be suggested that the PSEN-1-BID-16 complex may show relatively stable folded conformation during MD simulation. In case of PSEN-2, the same pattern was followed, where the Rg plot of protein-BID-16 was found to be stable in comparison with that of the protein-BID-20 complex. The Figs 9D and 10D shows the graphical plot of solvent accessible surface area (SASA) through which conformational changes between the interaction is analysed. The plot was evaluated based on the SASA values with simulation time i.e. 100 ns for both the modelled protein with BID-16. The average value of PSEN-1 with BID-16 was 181 nm2. Whereas, the protein-BID-19 complex was found to in the same pattern. In case of PSEN-2, both PSEN-2 bound BID-16 and BID-20 showed 160 nm2 of SASA value. Finally, the number of hydrogen bonds which was formed during the interaction were calculated. In this analysis, BID-16 showed up to 8 hydrogens bonds, whereas BID-19 showed only 3 of them  were found with more standard deviations. The higher standard deviations depict the inconsistent and unstable binding interactions during molecular dynamics simulation.Evaluation of binding free energy was done using MM-PBSA, which is the summation of non-polar, polar and non-bonded interaction energies. The summary of the free binding free energy calculations of BID-16 and controls (BID-19 and BID-20) bound to PSEN-1 and PSEN-2 have been given in , 25. Accumulation of Aβ peptide primarily due to the increased level of AChE effects the plaques deposition leading to the pathological condition . So far, there are several classes of AChE inhibitors, some of which are said to be FAD approved drugs commercially available. These include donepezil, rivastigmine, and galantamine, which can only slow the progression of AD but cannot prevent or completely treat the condition.Benzophenone derivatives are known to be promising treatment for AD because of their acetylcholinesterase (AChE) inhibiting potential, which helps in increasing the level of the neurotransmitter actions in-silico approach to investigate the effect of benzophenone based compounds out of which BID-16 was chosen after the virtual screening against PSEN-1 and PSEN-2 targets to know the effect on AD and to evaluate the biological activity, pharmacological effect, binding interaction, and dynamic nature on the target proteins. To analyse the molecular interaction of the compounds with modelled PSEN-1 and PSEN-2 proteins, docking study was conducted, after which the compound BID-16 was chosen for further investigation as it showed better binding affinity of -10.2 kcal/mol for PSEN-1 and -9.4 kcal/mol for PSEN-2, which were considered as the lowest negative values. The lowest negative values indicate the better binding affinity and interaction. To better understand the interaction patterns, the Discovery Studio Visualizer 2021 was used to analyze the docked conformations. The evaluation of PSEN-1 and PSEN-2 bound to BID-16 showed that it is bound within the binding site of both the proteins.In this study, we use in silico. The UniProt sequence and binding pocket analysis of PSEN-1 depict that 2 of the catalytic residues (ASP B: 257 and ASP B: 385) are involved in the inactivation of the enzyme activity, if the inhibitor binds to them , 27. Since PSEN-2 being a functionally similar and homologous to PSEN-1 , binding of inhibitor molecules to the binding pocket could induce the protein inactivity.The binding interaction of BID’s with both PSEN-1 and PSEN-2 have been analysed Yang et al. (2021) states that both PSEN-1 and PSEN-2 are the fuctional subunits of γ-secretase (PDB ID: 7C91). The study also states that ASP B: 257 and ASP B: 385 are the catalytic residues of the PSEN-1 present in the γ-secretase. When they investigated the inhibition activity of avagacestat, the compound bound to LEU B: 268 . In our study, BID-16 was able to bind with both ASP B: 385 and LEU B: 268 . With these studies, we can conclude that BID-16 could be a potent inhibitor of PSEN-1.A study by . When we performed molecular docking simulation, LY450139 was able to bind with our homology built models of PSEN-1  and PSEN-2 in silico. The docking results depict that ALA 415 of PSEN-2 is a common binding residue of LY450139 and BID-16. The LY450139 and BID-16 were able to bind with GLY B: 384  and ASP 385  of PSEN-1, respectively. The binding interactions of LY450139 have been depicted in S2 Table. Moreover, our study highlights the binding of BID-16 to the same binding pocket of the PSEN-1 and PSEN-2 that has been highlighted in the above mentioned studies. With these outcomes, we can conclude that BID’s can act the potential inhibitors of both the presenilin proteins.However, another γ-secretase inhibitor drug known as LY450139, is in Phase III clinical trial The BID-16 was taken for further investigation to assess its druglikeliness and pharmacokinetics properties, and the total binding energy formed during the dynamic simulation. The drug score of BID-16 was found to be 0.76. The compound passed the Lipinski’s rule of five, as it had all the essential properties. Likewise, there was no liver cytotoxicity, which is one of the most important parameter for drugs suitability evaluation. The compound demonstrated no Cytochrome P and p-gp inhibition, suggesting its potential as a promising drug candidate. Therefore, BID-16 can be considered to have better oral absorption.Furthermore, the molecular dynamic study was conducted to know the stability and structural changes of the protein-ligand complexes. In this study, we performed a MDS run for 100 ns for the complexes along with its reference compounds. The RMSD, RMSF, Rg, SASA and ligand hydrogen bonds were analysed using MD trajectories. All the results from MD analysis depict BID-16 as a promising drug candidate in terms of its stability and extensive binding interaction inside the binding pocket of the proteins for 100 ns. However, the changes observed in the trajectory plots reveal that interaction of BID-16 with both the proteins has been exceptional in terms of overall stability, in comparison with the negative controls. The values of RMSD, RMSF, Rg, SASA and ligand hydrogen bonds for protein-BID-16 complex are in accordance with those of protein backbone atoms. However, proteins complexed with negative controls showed inferior results. In addition, the binding free energy calculations showed that BID-16 bound PSEN-1 and PSEN-2 had lower binding free energies, indicating that the binding efficiency improved over the time frame. Also, standard deviations depict that the molecular dynamics simulation done for the protein-BID-16 has been remarkable in terms its stability, in comparison with the negative controls.in silico approach. Till date, there have been no studies in this aspect. So far, this is the first computational investigation on PSEN-1 and PSEN-2 proteins, which provides an insight into the mechanism of the inhibition by BID’s.The hallmark of this study is to investigate the presenilin inhibitory potential of BID’s through in vitro and in vivo aspects to prove its potential as an anti-Alzheimer’s drug.The main framework of this study was to understand the interaction of benzophenone integrated derivatives with presenilin (PSEN) proteins using computational tools. The molecular docking and interaction study showed that the compound BID-16 demonstrated better binding affinity for both presenilin (PSEN) proteins. Further, pharmacological potential and toxicity analysis was conducted, which screened the BIDs and predicted BID-16 as the most potent inhibitor. Finally, molecular dynamics showed that BID-16 is stable and has better affinity inside the binding pocket of both the proteins. To get a better understanding of the free energy formed during the simulation, binding free energy were calculated, which depicted the role of Van der Waal’s and binding energies in the formation of PSEN and BID-16 complexes. Based on these results, it can be deduced that BID-16 is a potential drug candidate for presenilin protein activity inhibition. However, further validation of BID-16 is needed with respect to S1 Fig(DOCX)Click here for additional data file.S2 Fig(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file. 13 Dec 2021 <div> PONE-D-21-34824 </div>  <div> Discovery of novel benzophenone integrated derivatives as anti-Alzheimer’s agents targeting presenilin-1 and presenilin-2 inhibition: a computational approach </div>  <div> PLOS ONEDear Dr. Ramu,Thank you for submitting your manuscript to PLOS ONE. 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Is the manuscript technically sound, and do the data support the conclusions? </font> The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: PartlyReviewer #2: Partly********** <font color="black"> 2. Has the statistical analysis been performed appropriately and rigorously?  </font> Reviewer #1: NoReviewer #2: Yes********** <font color="black"> 3. Have the authors made all data underlying the findings in their manuscript fully available?PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). 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You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. Reviewer #1: The study analyzed the binding affinity between benzophenones and PSEN-1/2 for demonstrating the pharmacotherapeutic potential by inhibiting PSEN-1/2. However, the study only focused on analyzing the affinities and interactions, without further investigation of the preferential binding pocket for the inhibition effect. Herein, it is hard to conclude the inhibition efficiency of screened benzophenone from these preliminary data, and many issues need to be addressed as followings:1. Figure 3-4, the binding pocket and binding sequence labeled in different colors should be clearly explained in the figure caption.2. Table 3-4, the in parallel comparison of binding affinity and non-bonding interactions of various BIDs are encouraged to present in column figures for better illustration. Also, please highlight the molecule structural differences of these BIDs in different color.3. Figure 5-6, it is hard to distinguish between the Pi-alkyl and alkyl interactions.4. The binding interactions between BIDs and PSEN-1/2 were analyzed in silico, but for the inhibition effect, how this binding contribute to the inhibition? Whether by stabilize the PSEN-1/2 conformation or block the active sites? All these need to be further introduced to demonstrate the inhibition benefits.5. In addition to the binding affinity, the binding pocket should also be analyzed for screening the best pose of BID-16 with PSEN-1/2.Reviewer #2: In the manuscript PONE-D-21-34824, the study evaluates the interaction of BIDs through molecular docking simulations, molecular dynamics simulations, and binding free energy calculations. Combinations of these approaches are acceptable. Although the manuscript is informative, there are still some questions that need to be addressed (see comments listed below).1. The false-positive rate of molecular docking is very high in the absence of experimental support. The literature listed by the author only mentions the inhibition of AChE/BACE-1 by Benzophenones, not PSEN-1/PSEN-2. Therefore, there is little significance in performing molecular docking study in such cases.doi.org/10.1021/ct300400x).2. The authors used CHARMM27 force field to get the parameter for protein (Line 126 to 129 in manuscript). However, their studies were based on an obsolete force field, which need a revision with the latest force fields. This can impact the results, weakening some analysis. One should use CHARMM36 instead of 27 . As a suggestion, the authors should use professional drawing software to improve the quality of these pictures.********** <font color="black"> 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Please note that Supporting Information files do not need this step.While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool,  18 Jan 2022Reviewer 1 commentsThe study analysed the binding affinity between benzophenones and PSEN-1/2 for demonstrating the pharmacotherapeutic potential by inhibiting PSEN-1/2. However, the study only focused on analysing the affinities and interactions, without further investigation of the preferential binding pocket for the inhibition effect. Herein, it is hard to conclude the inhibition efficiency of screened benzophenone from these preliminary data, and many issues need to be addressed as followings:1. Figure 3-4, the binding pocket and binding sequence labelled in different colours should be clearly explained in the figure caption.Response: As per the reviewer’s suggestion, binding pocket and binding sequence labelled in different colours (for both PSEN-1 and PSEN-2) have been explained in figure captions of Figure 3 (line 201-204) and Figure 4 (line 206-209).2. Table 3-4, the in parallel comparison of binding affinity and non-bonding interactions of various BIDs are encouraged to present in column figures for better illustration. Also, please highlight the molecule structural differences of these BIDs in different colour.Response: As per the reviewer’s suggestion, Table 3 and Table 4 have been merged. The merged table  now contains the structural differences of the BIDs highlighted in different colour. Also, binding affinity, total number of non-bonding interactions, total number of hydrogen bonds formed by BIDs during the docking simulation have also been given in adjacent columns. For the better illustration of these interaction parameters, Figure 5 and Figure 6 have been added, which represent the different docking parameters of PSEN-1 and PSEN-2, respectively.3. Figure 5-6, it is hard to distinguish between the Pi-alkyl and alkyl interactions.Response: As per the reviewer’s suggestion, colour of the Pi-alkyl interactions have been changed to dark pink colour . Also, row headings have been added in Table 4, which clearly describes the types of interactions to avoid confusion.4. The binding interactions between BIDs and PSEN-1/2 were analysed in silico, but for the inhibition effect, how this binding contributes to the inhibition? Whether by stabilize the PSEN-1/2 conformation or block the active sites? All these need to be further introduced to demonstrate the inhibition benefits. Response: As per the reviewer’s suggestion, in silico interaction of BIDs have now been supported with suitable information in “Discussion” section (lines 439-462) and supplementary data. In order to do this, authors have carefully performed the binding pocket analysis of both PSEN proteins, followed by the interaction analysis of BIDs with specific amino acid residues. We have identified a few key amino acid residues from the literature analysis, binding to which induces the inhibition activity. Binding interactions of BIDs also reveal that the key amino acids have been bound with the BIDs. Therefore, the interaction is oriented towards the inhibition of the protein activity by blocking the active binding residues.From previous studies conducted on γ-secretase, which consists of PSEN-1 and PSEN-2 proteins as functional subunits, we have provided the information of in silico inhibition of γ-secretase. The same inhibitor compound (LY450139) used in the study was docked with our homology model built PSEN-1 and PSEN-2 proteins. This has produced similar binding interactions like BIDs, which depicts that the binding interactions of BIDs have been oriented towards the inhibition, not the activation of the target proteins.5. In addition to the binding affinity, the binding pocket should also be analysed for screening the best pose of BID-16 with PSEN-1/2.Response: As per the reviewer’s suggestion, a thorough analysis of binding pockets of both PSEN-1 and PSEN-2 was conducted using the literature given in the UniProt database website. The literature survey resulted in the identification of few key amino residues, binding to which causes the inhibition of the protein activity. Results from our molecular docking studies reveal that BID-16 binds to the same amino acid residues, which shows that BID-16 as a potential inhibitor of both the PSEN-1 and PSEN-2 proteins. Difference in MD simulation plots were also given to highlight the impact of interaction (lines 476-481). During the virtual screening of the different binding poses, the first binding pose with zero RMSD and most negative binding affinity was selected. The same binding pose was found to interact with the key residues, in comparison with the other 9 binding poses.Reviewer 2 commentsIn the manuscript PONE-D-21-34824, the study evaluates the interaction of BIDs through molecular docking simulations, molecular dynamics simulations, and binding free energy calculations. Combinations of these approaches are acceptable. Although the manuscript is informative, there are still some questions that need to be addressed (see comments listed below).1. The false-positive rate of molecular docking is very high in the absence of experimental support. The literature listed by the author only mentions the inhibition of AChE/BACE-1 by Benzophenones, not PSEN-1/PSEN-2. Therefore, there is little significance in performing molecular docking study in such cases. Response: As per the reviewer’s suggestion, we conducted an extensive literature survey on the involvement of BIDs in the induction of anti-Alzheimer’s activity. However, there have been no studies performed in vitro for the inhibition of PSEN-1 and PSEN-2 by BIDs. The only available study on the anti-Alzheimer’s activity of BIDs is the inhibition of AChE/BACE-1 by Belluti et al. (2014) (reference no. 5). Therefore, we aimed to investigate the anti-Alzheimer’s activity through PSEN inhibition. This study provides an insight to the molecular mechanism of inactivating PSEN proteins through blocking their active sites. As this study is an in silico investigation, in vitro and in vivo studies in future could be performed using BIDs aiming for PSEN inhibition.doi.org/10.1021/ct300400x).2. The authors used CHARMM27 force field to get the parameter for protein (Line 126 to 129 in manuscript). However, their studies were based on an obsolete force field, which need a revision with the latest force fields. This can impact the results, weakening some analysis. One should use CHARMM36 instead of 27 . The table now contains data of structural differentiation of BIDs as well as their interaction details.5. One gets the impression that the work was assembled very hastily and without careful control. For instance, there are miscellaneous lines on the Fig.8B, which may be caused by the incorrect modification of the residue number. Also, Fig. 7B and 8B are missing ordinate labels. The units in the pictures should be unified . As a suggestion, the authors should use professional drawing software to improve the quality of these pictures.Response: As per the reviewer’s suggestion, negative controls were added for the MD simulation study. Using CHARMM36 forcefield, all the simulations were re-run for 100 ns. All the trajectories of MD simulations were plotted using XMGRACE. The plots were unified and put in one single picture using InkScape software.AttachmentAuthor Response (1).docxSubmitted filename: Click here for additional data file. 21 Feb 2022Discovery of novel benzophenone integrated derivatives as anti-Alzheimer’s agents targeting presenilin-1 and presenilin-2 inhibition: a computational approachPONE-D-21-34824R1Dear Dr. Ramu,We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. 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For more information, please contact Kind regards,Jie Zheng, Ph.DAcademic EditorPLOS ONEAdditional Editor Comments :Reviewers' comments: 22 Mar 2022PONE-D-21-34824R1 Discovery of novel benzophenone integrated derivatives as anti-Alzheimer’s agents targeting presenilin-1 and presenilin-2 inhibition: a computational approach Dear Dr. Ramu:I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. onepress@plos.org.If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact plosone@plos.org. 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Prolactin-secreting pituitary tumors (PRL-omas) are generally benign neoplasia. However, a percentage of cases show aggressive behavior. Prognostic markers may allow for the identification of aggressive cases. In this study, we investigated the prognostic role of galectin-3 and the estrogen receptor alpha (ERα), as predictive biomarkers of aggressiveness and poor prognosis.A mono-centric and retrospective study was conducted on consecutive cases of PRL-omas that underwent first line treatment with surgery and were followed-up for at least five years. The immunohistochemical expression of ERα and galectin-3 was investigated in each case.36 patients were enrolled. Galectin-3 resulted positive in 11 patients (30.6%). The median expression of ERα was 85% (IQR: 37). Among the group of 21 patients who underwent radical surgery (58.3%), recurrence occurred in 12 cases (33.3%). 27 patients were treated post-surgery with a dopamine agonist (DA) . 13 patients (48.1%) were responsive to DA. Six of 11 cases positive for galactin-3 underwent partial surgery . Recurrence occurred in all five cases that underwent radical surgery, which were also positive for galectin-3 (p=0.03). Galectin-3 resulted positive in 9 patients resistant to DA treatment . ERα expression was lower in tumors positive for galectin-3 (p<0.001), with mitotic activity (p=0.012), with higher Ki67 Li (p<0.001), and in males with post-surgical recurrence (p<0.001).Galectin-3 and ERα play as markers of aggressiveness and prognosis in PRL-omas and may be tested to identify the aggressive forms of the disease. Prolactin (PRL)-secreting pituitary adenomas are the most common subtype of pituitary tumors able to secrete hormones . The behUntil now male gender, invasiveness of para-sellar structures, and histopathological features (as a high proliferative index and an elevated mitotic count) were described as markers of aggressiveness in PRL-secreting tumors. The different gender-related expression of ERα has been hypothesized as involved in the clinical differences of the disease between females and males. However, previous studies and data remain discordant and not conclusive –8. RecenThe study aimed to evaluate the relationship between clinical features, tumor behavior, and the expression of galectin-3 and ERα, in a homogenous and retrospective surgical cohort of patients affected by prolactinoma with long-term follow-up.A mono-centric, retrospective, and interventional study was conducted on patients affected by PRL-secreting pituitary adenomas, consecutively enrolled, according to the following inclusion criteria:patients naïve to medical therapy with DA and underwent first line treatment with pituitary neurosurgery, conducted at our institution between 2005 and 2010;patients followed-up for at least five years, at our institution;patients who agreed to participate in the study, signing informed consent;available tumor specimens for interventional IHC evaluations for ERα, SSTR2, and galectin-3.Exclusion criteria. Patients were excluded from the study if:they underwent medical treatment with DA before pituitary surgery;the histological examination showed a pluri-hormonal IHC positivity of the pituitary adenoma.In our institution, also according to Klibanski , first-l-pituitary apoplexy;-cystic PRL-omas;-macro-PRL-omas in patients with a psychiatric condition for which DA is contraindicated;-after a joint and a multidisciplinary discussion between endocrinologists, neurosurgeons, and patients, concerning risks and benefits, with the final choice based upon the patient’s preference.Tumor specimens fixed in formalin and embedded in paraffin (FFPE) were retrieved from the archive of the Department of Pathology, Policlinico A. Gemelli IRCCS. In cases with multiple resections, we studied samples obtained at the time of the first surgery. Tumor specimens were processed according to standard protocols and stained with hematoxylin and eosin (HE). Immunostaining was performed on a Ventana Benchmark XT  using the following antibodies: anti-PRL , anti- Galectin 3 , anti-ERα , anti-SSTR2 , anti-p53  and anti-Ki67 . The mitotic rate was evaluated by the detection of mitosis on 10 HPF (High power field); the proliferation index was determined as a percentage of nuclear stained cells for KI-67 on at least two foci with the highest number of positive. p53 positivity scored as follows: negative, positive nuclei in < 5% of cells and positive nuclei in ≥ 5% of cells . The expression of ERα was evaluated as an absolute value.The main objective of the study was to investigate the prognostic role of galectin-3 and of the estrogen receptor alpha in predicting the outcome of PRL-secreting pituitary tumors.All patients underwent first-line neurosurgery. Pituitary surgery was considered subtotal in cases of persistence of hyperprolactinemia and tumor remnant at surgery and/or at MRI performed 3 months after surgery. Radical surgery was defined in cases of recovery of hyperprolactinemia and no evidence of tumor remnant at surgery and MRI performed 3 months after surgery. Recurrence was defined as hormonal and/or radiological disease reactivation during follow-up, in patients who had undergone tumor radical excision and with at least six months of disease-free survival. In fact, in our clinical practice, patients were followed-up according to a strict surveillance program. Hormonal dosage for prolactin and all the other pituitary hormones are conducted one and three months after pituitary surgery and then every six months, for the first five years. Similarly, pituitary MRI is performed six and twelve months after surgery and then annually. After five years of follow-up, cure pituitary hormonal tests are conducted annually and pituitary MR every two years.During the Al last evaluation available for this study, patients were considered cured if they underwent radical surgery and in absence of recurrence. Patients were considered to carry a persistent disease in cases of subtotal surgery or cases of recurrent disease. Disease status at the end of the study was defined as a measure of outcome. We classified patients as cured or with persistent disease at the end of follow-up.According to Melmed et al. , patient-partially resistant to DA in cases of decrease of the tumor size and prolactin levels without normalizing, requiring a higher dose of DA to achieve a complete response;-complete resistance to DA in cases of failure in obtaining normal prolactin values, and/or failure to reduce tumor size by 50% and/or failure to regain fertility with maximum tolerated doses of DA.The normal distribution of continuous variables was evaluated through the Kolmogorov-Smirnov test. Data were presented as median ± interquantile ranges (IQR) for continuous variables and as absolute and relative frequencies for qualitative variables. We performed univariate analysis for the evaluation of the relationship between the considered variables and disease outcome. Fisher exact test and non-parametric tests  were used to compare qualitative and continuous variables, respectively. We performed logistic regression and a survival analysis through Kaplan-Meier curve for the event “recurrence”. Statistical significance was assumed when p<0.05. Data were analyzed using the SSPS Software, version 23.The study was approved by the local ethics committee of Gemelli Hospital, Catholic University of the Sacred Heart.Thirty-six patients entered the study: 13 were women (36.1%) and 23 men (63.9%). The median age of diagnosis was 31 years (IQR: 17). The median duration of follow-up was 96 months (IQR: 56).Twenty-seven patients carried a macroprolactinoma , 7 patients a micro-prolactinoma  and 2 patients a giant prolactinoma, defined as larger than 4 cm in diameter (5.6%). Pituitary adenoma was invasive in 14 cases (38.9%): invasion of cavernous sinus occurred in 9 cases (five cases with grade 3 and four cases with grade 4 Knosp score), of dura meter in 2 cases, and sphenoid sinus in 3 cases. All patients underwent first line pituitary surgery. A radical surgical removal was reached in 21 cases and a subtotal removal in 15 cases. Recurrence occurred in 12 out of the 21 cases that underwent radical surgery. Treatment with DA was prescribed in 27 patients . Mean cabergoline dosage was 1 mg/weekly (minimum: 0.75 mg/weekly maximum 3 mg/weekly). 13 patients (48.1%) were responsive to DA and the remaining 14 (38.9%) were resistant. Three of the 14 DA resistant patients were treated with combination therapy with DA and SSA, reaching the disease control in 2 cases, in terms of normalization of the hyperprolactinemia and the tumor mass. At the end of the study, 9 patients (25%) were considered cured, 16 controlled through post-surgical medical therapy (41.7%), and 12 (33.3%) affected by persistent hyperprolactinemia, despite a high tolerated dose of DA.Galectin-3 resulted as positive in 11 patients (30.6%) and negative in 25 patients (69.4%). Data on galactin-3 expression in the study population are summarized in According to the patients’ prognosis Table 2,The median expression of ERα was 85% (IQR: 37). ERα expression was significantly lower in males as in females (p=0.037), in macro (p=0.003), and invasive (p<0.001) PRL-secreting tumors Figure 1As shown in The main determinants of resistance to DA were the Ki67≥ 1.5%  and the positivity of galectin-3 (p=0.03). In addition, we found that the expression of galectin-3 correlated with a shorter disease free survival p=0.02, Figure 2In this retrospective study, we investigated the prognostic role of galectin-3 and ERα, in predicting aggressiveness, post-surgical outcome, and responsiveness to DA therapy, in patients who underwent first line surgery for PRL-secreting pituitary tumors.The biological and clinical behavior of PRL-secreting pituitary tumors can be very variable, ranging from cases of micro- PRL-secreting pituitary tumors cured with DA to cases of aggressive tumors characterized by resistance to DAs, recurrence/progression, and at least cases of PRL-secreting pituitary carcinomas . RecentlOur study also revealed the role of ERα, as a predictor of aggressiveness and prognosis in PRL-secreting pituitary tumors. The expression of ERα in human lactotroph normal and tumoral cells was well established, although in absence of conclusive data on gender difference , 15–17. In a recent study conducted on 89 prolactinomas , the lowThe higher aggressiveness of PRL-secreting pituitary tumors in males is also well described by the clinico-pathological five-tiered classification of pituitary adenoma proposed by Trouillas et al. , as thesOther markers of aggressiveness in our series were the higher value of prolactin at diagnosis and the higher value of Ki67 that may predict the resistance to DA. As shown in previous papers , this stThe main limitations of our study are the small sample size and its retrospective design. However, this study described a monocentric series of patients affected by PRL-secreting pituitary tumors naïve to DA therapy and underwent first line treatment with pituitary surgery. At our institution, over the years, trans-sphenoidal pituitary surgery has been considered an important part of patient counseling in deciding upon an initial treatment, according to the low morbidity of this procedure, if performed by an experienced neurosurgeon . In partIn conclusion, although our results should be confirmed by a larger cohort and prospective studies, our results suggest that ERα and galectin-3 are markers of aggressiveness and prognosis in PRL-secreting pituitary tumors and may be tested for identifying aggressive forms of the disease, in the field of personalized therapy, with a multidisciplinary approach.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by Ethics committee of Fondazione Policlinico Gemelli, Rome. The patients/participants provided their written informed consent to participate in this study.CB and SC wrote the manuscript. CB performed the conceptualization, data curation, and data collection. SC performed the conceptualization and formal analysis. MG developed the methodology and conducted the histological investigation. AG, PM, LL, CA, GR, AP, LM, and AB conducted the validation of the research outputs. AP, LM, and AB performed the supervision of the research activities. All authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.